MHC class II (MHC-II) molecules associate with a chaperone protein termed the Invariant chain (Ii) that facilitates the transport of newly synthesized MHC-II to endo/lysosomal antigen processing compartments. Expression of Ii is required for APCs to stimulate most, but certainly not all, antigen-specific T cells. It is thought that these Ii-independent antigens are presented by pre-existing peptide-loaded MHC-II (pMHC-II) that internalize from the plasma membrane, enter into endosomal compartments, exchange old peptides for new antigenic peptides, and then recycle back to the plasma membrane for presentation to CD4 T cells. We have recently shown that unlike Ii-associated MHC-II, pre-existing pMHC-II on the plasma membrane rapidly internalizes into APCs and is returned to the plasma membrane following an Arf6, Rab35, and EHD1-dependent recycling pathway, thereby revealing the molecular machinery responsible for pMHC-II recycling in APCs. In most APCs, the endo/lysosomal antigen processing compartments have properties of multivesicular bodies (MVBs). MVBs are formed by the invagination of the limiting membrane of the organelle, giving rise to a &amp;#8776;500 nm structure filled with 50-100 nm vesicles. Curiously, MHC-II is present primarily on the intralumenal vesicles of MVB and it is thought that the sorting of MHC-II into these intralumenal vesicles is essential for proper MHC-II-peptide loading. The sorting signals that direct MHC-II onto the intralumenal vesicles of MVB have not been identified, and we will identify these signals and determine the extent to which specific recognition proteins (such as ESCRT), lipids (such as cholesterol or lysobisphosphatidic acid, LBPA), or post-translational modifications of MHC-II (such as ubiquitination) regulate MHC-II entry and/or exit from these compartments for antigen loading.MHC class II (MHC-II) molecules associate with a chaperone protein termed the Invariant chain (Ii) that facilitates the transport of newly synthesized MHC-II to endo/lysosomal antigen processing compartments. Expression of Ii is required for APCs to stimulate most, but certainly not all, antigen-specific T cells. It is thought that these Ii-independent antigens are presented by pre-existing peptide-loaded MHC-II (pMHC-II) that internalize from the plasma membrane, enter into endosomal compartments, exchange old peptides for new antigenic peptides, and then recycle back to the plasma membrane for presentation to CD4 T cells. We have recently shown that unlike Ii-associated MHC-II, pre-existing pMHC-II on the plasma membrane rapidly internalizes into APCs and is returned to the plasma membrane following an Arf6, Rab35, and EHD1-dependent recycling pathway, thereby revealing the molecular machinery responsible for pMHC-II recycling in APCs. In most APCs, the endo/lysosomal antigen processing compartments have properties of multivesicular bodies (MVBs). MVBs are formed by the invagination of the limiting membrane of the organelle, giving rise to a &amp;#8776;500 nm structure filled with 50-100 nm vesicles. Curiously, MHC-II is present primarily on the intralumenal vesicles of MVB and it is thought that the sorting of MHC-II into these intralumenal vesicles is essential for proper MHC-II-peptide loading. The sorting signals that direct MHC-II onto the intralumenal vesicles of MVB have not been identified, and we will identify these signals and determine the extent to which specific recognition proteins (such as ESCRT), lipids (such as cholesterol or lysobisphosphatidic acid, LBPA), or post-translational modifications of MHC-II (such as ubiquitination) regulate MHC-II entry and/or exit from these compartments for antigen loading.